Inhibition of JNK Signaling Overcomes Cancer-Associated Fibroblast-Mediated Immunosuppression and Enhances the Efficacy of Immunotherapy in Bladder Cancer [scRNA-Seq]

Currently, only 20-40% of cancer patients benefit from immune checkpoint inhibitors. Understanding the mechanisms underlying the immunosuppressive tumor microenvironment (TME) and characterizing dynamic changes in the immunological landscape during treatment are critical for improving responsiveness to immunotherapy. Here, we identified JNK signaling in cancer-associated fibroblasts (CAFs) as a regulator of the immunosuppressive tumor microenvironment. Single-cell RNA sequencing of bladder cancer treated with a JNK inhibitor revealed enhanced cytotoxicity and effector functions of CD8+ T cells. In untreated tumors, CAFs interacted frequently with CD8+ T cells and mediated their exhaustion. JNK inhibition abrogated the immunosuppression function of CAFs by downregulating the expression of TSLP, thereby restoring CD8+ T cell cytotoxicity. In addition, blockade of CAF-derived TSLP in combination with anti-PD1 treatment promoted tumor elimination by CD8+ T cells in vivo. Collectively, these results indicate that JNK signaling plays an important immunosuppressive role in the tumor microenvironment by promoting expression of TSLP in CAFs and suggest that inhibiting JNK signaling could be a promising immunotherapeutic strategy for cancer treatment. To systematically investigate the cellular diversity and molecular variation in the bladder tumor microenvironment following JNK inhibition, we collected whole bladders from N-Butyl-N(4-hydroxybutyl) nitrosamine-induced mice (2 samples per group) and performed single-cell RNA sequencing using 10x genomics platform.

目前仅有20%~40%的癌症患者可从免疫检查点抑制剂（immune checkpoint inhibitors）中获益。阐明免疫抑制性肿瘤微环境（tumor microenvironment, TME）的调控机制，并解析治疗过程中免疫景观的动态变化，对提升免疫治疗应答率至关重要。本研究鉴定出癌症相关成纤维细胞（cancer-associated fibroblasts, CAFs）中的JNK信号通路作为免疫抑制性肿瘤微环境的调控因子。对经JNK抑制剂（JNK inhibitor）处理的膀胱癌样本开展单细胞RNA测序（single-cell RNA sequencing）分析后发现，CD8+ T细胞的细胞毒性与效应功能显著增强。在未接受治疗的肿瘤中，CAFs可与CD8+ T细胞频繁发生互作，并介导后者的耗竭。JNK抑制剂可通过下调胸腺基质淋巴细胞生成素（thymic stromal lymphopoietin, TSLP）的表达，消除CAFs的免疫抑制功能，从而恢复CD8+ T细胞的细胞毒性。此外，在体内联合阻断CAFs来源的TSLP与抗PD-1（anti-PD1）治疗，可促进CD8+ T细胞清除肿瘤组织。综上，本研究结果表明，JNK信号通路可通过促进CAFs中TSLP的表达，在肿瘤微环境中发挥关键的免疫抑制作用，提示抑制JNK信号通路有望成为一种极具前景的癌症免疫治疗策略。为系统探究JNK抑制后膀胱癌肿瘤微环境的细胞异质性与分子变异，我们收集了N-丁基-N-(4-羟丁基)亚硝胺（N-Butyl-N(4-hydroxybutyl) nitrosamine）诱导的小鼠全膀胱组织（每组2个样本），并采用10x Genomics平台完成单细胞RNA测序。